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Dive into the research topics where Lucia Daddabbo is active.

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Featured researches published by Lucia Daddabbo.


Journal of Biological Chemistry | 2012

Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

Magnus Monné; Daniela Valeria Miniero; Lucia Daddabbo; Alan J. Robinson; Edmund R. S. Kunji; Ferdinando Palmieri

Background: Substrate binding and transport mechanisms of mitochondrial carriers are inadequately understood. Results: The effect of mutations on substrate specificity and transport activity was assessed in two human ornithine carrier isoforms. Conclusion: The substrate specificity and transport rate of the two isoforms are defined by a few residues and can be swapped. Significance: The results show how small substrates can trigger transport in carriers. Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the Cα carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.


Amino Acids | 2015

Mitochondrial transporters for ornithine and related amino acids: a review

Magnus Monné; Daniela Valeria Miniero; Lucia Daddabbo; Luigi Palmieri; Vito Porcelli; Ferdinando Palmieri

Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to μM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast.


Biochimica et Biophysica Acta | 2011

Functional and structural role of amino acid residues in the matrix α-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier

Daniela Valeria Miniero; Anna Rita Cappello; Rosita Curcio; Anna Ludovico; Lucia Daddabbo; Italo Stipani; Alan J. Robinson; Edmund R. S. Kunji; Ferdinando Palmieri

The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.


Journal of Bioenergetics and Biomembranes | 1999

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

Dorotea Natuzzi; Lucia Daddabbo; Valentina Stipani; Anna R. Cappello; Daniela Valeria Miniero; Loredana Capobianco; Italo Stipani

The effect of pyridoxal 5′-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5′-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiffs base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5′-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [3H]pyridoxal 5′-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5′-phosphate. These results indicate that pyridoxal 5′-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.


Biochimica et Biophysica Acta | 1995

Photoaffinity labeling of the mitochondrial oxoglutarate carrier by azido-phthalonate

Italo Stipani; Dorotea Natuzzi; Lucia Daddabbo; Alberto Ritieni; Giacomino Randazzo; Ferdinando Palmieri

The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.


Journal of Biological Chemistry | 2018

Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates

Magnus Monné; Lucia Daddabbo; David Gagneul; Toshihiro Obata; Björn Hielscher; Luigi Palmieri; Daniela Valeria Miniero; Alisdair R. Fernie; Andreas P. M. Weber; Ferdinando Palmieri

The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides, and cofactors across the mitochondrial membrane. Here, two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified, and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteine sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate, and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. AtUCP1 and AtUCP2 catalyzed a fast counterexchange transport as well as a low uniport of substrates, with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts shows changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartateout/glutamatein exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.


Journal of Molecular Biology | 2006

Functional and Structural Role of Amino Acid Residues in the Even-numbered Transmembrane α-Helices of the Bovine Mitochondrial Oxoglutarate Carrier

Anna Rita Cappello; Daniela Valeria Miniero; Rosita Curcio; Anna Ludovico; Lucia Daddabbo; Italo Stipani; Alan J. Robinson; Edmund R. S. Kunji; Ferdinando Palmieri


Biochemistry | 2001

The mitochondrial oxoglutarate carrier: Cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents

Valentina Stipani; Anna Rita Cappello; Lucia Daddabbo; Dorotea Natuzzi; Daniela Valeria Miniero; Italo Stipani; Ferdinando Palmieri


Journal of Biological Chemistry | 1995

Identification by in Organello Footprinting of Protein Contact Sites and of Single-stranded DNA Sequences in the Regulatory Region of Rat Mitochondrial DNA

Lucia Daddabbo


Biochimica et Biophysica Acta | 2015

Functional characterization and organ distribution of three mitochondrial ATP–Mg/Pi carriers in Arabidopsis thaliana

Magnus Monné; Daniela Valeria Miniero; Toshihiro Obata; Lucia Daddabbo; Luigi Palmieri; Angelo Vozza; M. Cristina Nicolardi; Alisdair R. Fernie; Ferdinando Palmieri

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Alan J. Robinson

MRC Mitochondrial Biology Unit

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